Use of an antagonist of PPARα and PPARγ for the treatment of syndrome X

ABSTRACT

A method for the treatment or prophylaxis of Syndrome X in a human or non-human mammal is disclosed. The method comprises the administration of an effective, non-toxic and pharmaceutically effective amount of an agonist of PPARα and PPARγ, or a pharmaceutically acceptable derivative thereof, to a human or non-human mammal in need thereof.

This application is a 371 of PCT/EP97/00358, filed Jan. 7, 1997.

This invention relates to a novel method of treatment, in particular forthe treatment of Syndrome X and certain compounds used in said method.

It is known that the γ-isoform of peroxisome proliferator-activatedreceptor (herein after PPARγ) is member of a nuclear receptorsuperfamily that includes receptors for the steroid, thyroid andretinoid hormones(Evans, Science 240, 889-895, (1988)). It is also knownfrom Chawla et al that PPARγ is expressed early during thedifferentiation of adipocytes (Endocrinology 135,798-800, 1994).

Spiegelman et al state in Cell (Vol 83, 803-812, 1995) that signalswhich modulate PPARγ activity may serve a primary role in regulatingenergy homeostasis. They conclude (ibid, 810) that `screening for potentPPARγ agonists and antagonists represents a logical and potentiallyrapid approach towards the development of novel therapeutic agents forNIDDM and obesity respectively.`.

It is known that the α-isoform of peroxisome proliferator-activatedreceptor (herein after PPARα) acts to stimulate peroxisomalproliferation in the rodent liver which leads to enhanced fattyoxidation by this organ (Keller and Wahli: Trends Endocrin Metab1993;4:291-296). Hypolipidaemic agents have the ability to stimulatePPAR alpha and the ensuing stimulation of peroxisomal proliferation andconsequent fatty acid oxidation can account for the reduction in plasmafatty acids (Macdonald and Lane: Current Biology Vol5 pp618-621 (1995)).

Syndrome X is the syndrome characterised by an initial insulin resistantstate, generating hyperinsulinaemia, dyslipidaemia and impaired glucosetolerance, which can progress to non-insulin dependent diabetes mellitus(Type II diabetes), characterised by hyperglycaemia and which thenfurther progresses to diabetic complications.

We now consider that inclusion of PPARα effects in a PPARγantihyperglycaemic agent will result in a reagent with enhancedtherapeutic potential in the syndrome X aetiology due to an enhancedhypolipidaemic effect.

International Patent Application number PCT/EP 95/03038 disclosescertain compounds of formula (A): ##STR1## or a pharmaceuticallyacceptable salt thereof, and/or a pharmaceutically acceptable solvatethereof, wherein R^(a) represents 2-benzoxazolyl or 2-pyridyl and R^(b)represents CH₂ OCH₃ or CF₃.

The compounds of formula (A) are stated to be of potential use in thetreatment and/or prophylaxis of hyperglycaemia, especially in Type IIdiabetes, hyperlipidaemia, hypertension, cardiovascular disease,especially atherosclerosis and of renal disease, especially renaldisease associated with the development of Type II diabetes includingdiabetic nephropathy, glomerulonephritis, glomerular sclerosis,nephrotic syndrome, hypertensive nephrosclerosis and end stage renaldisease and for the prevention, reversal, stabilisation or retardationof the progression of microalbuminuria to albuminuria.

It has now been discovered that the compounds of formula (A) exhibitagonist activity at both PPARα and PPARγ and that as well as beingparticularly effective for the treatment and/or prophylaxis ofhyperglycaemia they are also now considered to be most effective for thetreatment and/or prophylaxis of pre-diabetic insulin resistance syndromeand the resulting complications thereof: They are therefore consideredto be useful for the treatment and/or prophylaxis of insulin resistance,diabetes, dyslipidaemia, atherosclerosis, hypertension, cardiovasculardisease and obesity.

Accordingly, in a first aspect the invention provides a method for thetreatment and/or prophylaxis of Syndrome X in a human or non-humanmammal, which method comprises the administration of an effective,non-toxic and pharmaceutically effective amount of an agonist of PPARαand PPARγ, or a pharmaceutically acceptable derivative thereof, to ahuman or non-human mammal in need of such treatment.

Thus, one aspect of the invention is the treatment of Syndrome X.

A further aspect of the invention is the prophylaxis of Syndrome X.

In particular, there is provided a method for the treatment and/orprophylaxis of hyperglycaemia.

In particular there is provided a method for the treatment ofpre-diabetic insulin resistance syndrome and the resulting complicationsthereof.

Pre-diabetic insulin resistance syndrome includes hyperinsulinaemia andimpaired glucose tolerance.

In a further aspect, there is provided a method for the treatment and/orprophylaxis of Syndrome X, including hyperglycaemia and/or pre-diabeticinsulin resistance syndrome and the resulting complications thereof, ina human or non-human mammal, which method comprises the administrationof an effective, non-toxic and pharmaceutically effective amount of anagonist of PPARα and PPARγ, or a pharmaceutically acceptable derivativethereof, to a human or non-human mammal in need thereof; providing themethod does not include the administration of:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid, for the treatment and/or prophylaxis of: hyperglycaemia,especially in Type II diabetes, hyperlipidaemia, hypertension,cardiovascular disease, especially atherosclerosis and of renal disease,especially renal disease associated with the development of Type IIdiabetes including diabetic nephropathy, glomerulonephritis, glomerularsclerosis, nephrotic syndrome, hypertensive nephrosclerosis and endstage renal disease and for the prevention, reversal, stabilisation orretardation of the progression of microalbuminuria to albuminuria.

It will be appreciated that in its preferred form the agonist of PPARαand PPARγ is a single compound (such compound being referred to hereinas a `PPARα and γ agonist`) but it is within the ambit of this inventionthat the agonist of PPARα and PPARγ is provided by a combination of aPPARα agonist compound and a PPARγ agonist compound.

One combined PPARα and γ agonist is a compound of formula (I): ##STR2##or a pharmaceutically acceptable salt thereof, and/or a pharmaceuticallyacceptable solvate thereof, wherein R⁰ represents 2-benzoxazolyl or2-pyridyl and R¹ represents CH₂ OCH₃ or CF₃.

Preferably, R⁰ represents 2-benzoxazolyl.

Suitably, R¹ represents CH₂ OCH₃.

Preferably, R¹ represents CF₃.

The compounds of formula (I), and the pharmaceutically acceptable saltsthereof, may exist in one of several tautomeric forms, all of which areencompassed by the present invention as individual tautomeric forms oras mixtures thereof.

Pharmaceutically acceptable derivatives include pharmaceuticallyacceptable salts and solvates.

Suitable pharmaceutically acceptable salts include salts of carboxygroups and acid addition salts.

Suitable pharmaceutically acceptable salts of carboxy groups includemetal salts, such as for example aluminium, alkali metal salts such aslithium, sodium or potassium, alkaline earth metal salts such as calciumor magnesium and ammonium or substituted ammonium salts, for examplethose with lower alkylamines such as triethylamine, hydroxy alkylaminessuch as 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine, cycloalkylamines such as bicyclohexylamine,or with procaine, dibenzylpiperidine, N-benzyl-β-phenethylamine,dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine,N-methylglucamine or bases of the pyridine type such as pyridine,collidine, quinine or quinoline.

Suitable acid addition salts include pharmaceutically acceptableinorganic salts such as the sulphate, nitrate, phosphate, borate,hydrochloride and hydrobromide and, where feasible, pharmaceuticallyacceptable organic acid addition salts such as acetate, tartrate,maleate, citrate, succinate, benzoate, ascorbate, methanesulphonate,α-keto glutarate and α-glycerophosphate.

Suitable pharmaceutically acceptable solvates include hydrates.

The pharmaceutically acceptable derivatives, such as the salts and/orsolvates of the compounds of formula (I) may be prepared and isolatedaccording to conventional procedures, for example sodium salts may beprepared by using sodium methoxide in methanol.

A compound of formula (I), or a pharmaceutically acceptable saltthereof, and/or a pharmaceutically acceptable hydrate thereof, may beprepared by hydrolysing a compound of formula (II): ##STR3## wherein R⁰and R¹ are as defined in relation to formula (I) and L¹ represents ahydrolysable group; and thereafter, if required, preparing apharmaceutically acceptable salt of the compound of formula (I) and/or apharmaceutically acceptable solvate thereof.

A suitable hydrolysable group L¹ is a group of formula (a) or an epimerthereof: ##STR4##

A suitable hydrolysable group L¹ is an Evans chiral auxiliary, forexample a group of formula (b) or an epimer thereof: ##STR5##

A suitable hydrolysable group L¹ is a C₁₋₆ alkoxy group.

The hydrolysis of the compound of formula (II) is carried out usingconditions appropriate for hydrolysing the particular group L¹ chosen,for example when L¹ is a group of formula (a) or a C₁₋₆ alkoxy group,the hydrolysis is suitably carried out under acidic conditions, forexample using dilute sulphuric acid, conveniently in a water/dioxanmixture, for example a 1:1 mixture, at any temperature which provides asuitable rate of formation of the required product, generally at anelevated temperature, such as in the range of from 50° C. to 120° C.,for example 90° C.; or when L¹ is a group of formula (b) the hydrolysisis generally carried out using lithium hydroperoxide in an aqueoussolvent, such as aqueous tetrahydrofuran, at any temperature whichprovides a suitable rate of formation of the required product, generallyat a reduced temperature, such as in the range of from -10° C. to 0° C.,for example 0° C. Alternatively, when L¹ is a group of formula (b) thehydrolysis may be effected under basic conditions, using for exampleaqueous sodium hydroxide, in an appropriate solvent such as aqueoustetrahydrofuran usually at ambient temperature.

A compound of formula (II), wherein L¹ is a moiety of the above definedformula (a) or (b), may be prepared from a compound of formula (III):##STR6## wherein R⁰ and R¹ are as defined in relation to formula (I) andL² represents a leaving group; (i) for compounds of formula (II) whereinL¹ is a moiety of the above defined formula (a), by reaction with(S)-phenylglycinol; or (ii) for compounds of formula (II) wherein L¹ isa moiety of the above defined formula (b), by reaction with(S)-4-benzyloxazolidin-2-one, preferably an activated form thereof; andthereafter separating the required isomer from the mixture ofdiastereoisomers produced.

A suitable leaving group L² is a halogen atom, for example a chlorineatom.

The reaction between the compounds of formula (III) and(S)-phenylglycinol may be carried out under conventional amidationconditions, for example in an inert solvent such as dichloromethane at atemperature which provides a suitable rate of formation of the requiredproduct, suitably at ambient temperature and preferably in the presenceof a base such as triethylamine.

A suitable activated form of (S)-4-benzyloxazolidin-2-one is a saltedform, for example an alkali metal salted form, preferably a lithiumsalt.

The activated form of (S)-4-benzyloxazolidin-2-one may be prepared byany appropiate conventional method. Thus when the activated form is alithium salt, it may be prepared by treating(S)-4-benzyloxazolidin-2-one with a source of lithium ions in thepresence of a base, suitably provided by n-butyllithium, in an aproticsolvent such as tetrahydrofuran, usually at a low temperature, forexample in the range of from -78° to 0° C.

The reaction between the compound of formula (III) and the activatedform of (S)-4-benzyloxazolidin-2-one may be carried out in an aproticsolvent, such as tetrahydrofuran, at a temperature which provides asuitable rate of formation of the required product, conveniently byallowing the reaction mixture to slowly warm from -78° to 0° C.

Preferably, the activated form of (S)-4-benxyloxazolidin-2-one isprepared and then reacted in-situ with the compound of formula (III).

A compound of formula (III) may be prepared by hydrolysing thecarboxylic ester COOR² of a compound of formula (IV): ##STR7## whereinR⁰ and R¹ are as defined in relation to formula (I) and R² represents analkyl group, and thereafter converting the carboxylic acid group soformed into a moiety CO.L².

A suitable alkyl group R² is a C₁₋₆ alkyl group, especially a methylgroup.

The hydrolysis of the carboxylic ester may be effected by use of anyconventional hydrolysing agent, such as an alkaline metal hydroxide, forexample sodium hydroxide.

The hydrolysis of the compound of formula (IV) may be carried out in anysuitable solvent such as a methanol/water mixture, conveniently a 1:1mixture, at a temperature which provides a suitable rate of formation ofthe required product, suitably at an elevated temperature andconveniently at the reflux temperature of the solvent.

The conversion of the carboxylic acid group into the moiety CO.L² may becarried out using any appropiate conventional procedure, depending uponthe particular nature of the group L² chosen, thus when L² is a halogena suitable procedure involves treatment of the carboxylic acid with anoxalyl halide, for example oxalyl chloride when L² is chlorine.

The reaction conditions for the conversion of the carboxylic acid groupinto the moiety CO.L² will be dictated by the particular nature of L²and the source of L² chosen, for example when L² is halogen and thesource of L² is oxalyl chloride then the reaction may be carried out inan inert solvent such as dichloromethane or benzene at a temperaturewhich provides a suitable rate of formation of the required product,suitably at ambient temperature or at an elevated temperature such asthe reflux temperature of the solvent.

It will be appreciated that the preparation and separation of a compoundof formula (II) wherein L¹ is an epimer of the above defined moiety (a)or (b) and its subsequent hydrolysis to afford a compound of formula (I)can be achieved by employing analogous methods to those described abovefor the preparation, separation and hydrolysis of a compound of formula(II) wherein L¹ represents the above defined moiety (a) or (b).

A compound of formula (II) wherein L¹ is a moiety of formula (b) mayalso be prepared by dehydroxylation of a compound of formula (V):##STR8## wherein R⁰ and R¹ are as defined in relation to formula (I) andX is a moiety of the above defined formula (b).

The dehydroxylation of the compound of formula (V) is convenientlycarried out by treatment with a trialkylsilane, for exampletriethylsilane, preferably in the presence of trifluoroacetic acid andconveniently using trifluoroacetic acid as solvent, at any temperatureproviding a suitable rate of formulation of the product, for example ata temperature in the range from 0° C. to room temperature.

It will be appreciated that a compound of formula (II) wherein L¹ is amoiety of formula (b) would also be obtained by dehydroxylation of acompound of formula (V) in which the hydroxy bearing stereocentre isepimerised.

A compound of formula (V) may be prepared by reacting a compound offormula (VIA): ##STR9## wherein R⁰ is as defined in relation to formula(I), with a compound of formula (VIB): ##STR10## wherein R¹ is asdefined in relation to formula (I); and thereafter separating therequired isomer from the mixture of diastereoisomers produced.

Suitably in the above mentioned reaction, the compound of formula (VIB)is in an activated form, which is preferably provided by treating thecompound of formula (VIB) with an alkylboron triflate, for exampledibutylboron triflate, preferably in the presence of an amine base suchas triethylamine.

The activated form of the compound of formula (VIB) may be prepared bythe appropriate conventional method depending upon the specific natureof the activated form chosen, for example the compound of formula (VIB)is reacted with dibutylboron triflate and triethylamine in an inertsolvent such as dichloromethane at a temperature in the range of from-78° to 0° C.

The reaction between the compounds of formulae (VIA) and (VIB) may becarried out in an in an inert solvent such as dichloromethane, at atemperature which provides a suitable rate of formation of the requiredproduct, conveniently by allowing the reaction mixture to slowly warmfrom -78° to 0° C.

Preferably, the activated form of the compound of formula (VIB) isprepared and then reacted in-situ with the compound of formula (VIA).

For compounds of formula (I) wherein R⁰ represents 2-benzoxazolyl, asuitable compound of formula (VIA) is4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]benzaldehyde.

A suitable means for separating any required single isomer from amixture of diastereoisomers is chromatography, such as preparative highpressure liquid chromatography or silica gel column chromatography.

One convenient method for preparing a compound of formula (II) whereinL¹ is a C₁₋₆ alkoxy group is the basic alcoholysis of a compound offormula (II) wherein L¹ is a moiety of formula (b).

A suitable base is an alkali metal alkoxide, for example when L¹ ismethoxy the compound of formula (II) wherein L¹ is moiety (b) is treatedwith sodium methoxide in methanol.

A compound of formula (I) may also be prepared by resolving a racemiccompound of formula (VII): ##STR11## wherein R⁰ and RI are as defined inrelation to formula (1); and thereafter, if required, preparing apharmaceutically acceptable salt of the compound of formula (I) and/or apharmaceutically acceptable solvate thereof.

The resolution of a compound of formula (VII) may be carried out usingknown resolution procedures, for example by reacting the compound offormula (VII) with a resolving agent, such as an optically active acidor base, to provide a mixture of diastereoisomeric salts which may thenbe separated by fractional crystallisation and thereafter the compoundof formula (I) may be regenerated from the separated diastereoisomersalt by conventional means, such as hydrolysis.

It will be appreciated that the compounds of formula (VII) comprise thecompounds of formula (I) admixed with other optical isomers. A compoundof formula (VII) or a pharmaceutically acceptable salt thereof and/or apharmaceutically acceptable solvate thereof, forms a further aspect ofthe present invention. The separated isomers of the compounds of formula(VII), in addition to the compounds of formula (I), or apharmaceutically acceptable salt thereof and/or a pharmaceuticallyacceptable solvate thereof, also comprise the present invention.

Suitable acids or bases for resolving the compounds of formula (VII) areas described in Enantiomers, Racemates and Resolution, J Jaques et al,1981, Wiley Interscience, especially at pages 255 and 256. Suitablemethods for effecting the resolution are also disclosed by Jaques et al.

The compounds of formula (IV) and (VIA), for example4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]benzaldehyde, are knowncompounds or they may be prepared using methods analogous to those usedto prepare known compounds, for example those disclosed in InternationalPatent Application, Publication Number WO94/01420.

The compounds of formula (VIB) are known compounds or they may beprepared using methods analogous to those used to prepare knowncompounds, for example those disclosed in Organic Synthesis Vol. 68,p83, 1990 Ed. J. D. White or methods analogous thereto, in combinationwith conventional methodology for the preparation of acid chlorides.

It will be appreciated that in any of the abovementioned reactions anyreactive group in the substrate molecule may be protected, according toconventional chemical practice. Suitable protecting groups in any of theabovementioned reactions are those used conventionally in the art. Themethods of formation and removal of such protecting groups are thoseconventional methods appropriate to the molecule being protected.

It will be appreciated that the above mentioned preparation of thecompounds of formula (I), or a pharmaceutically acceptable salt thereofand/or a pharmaceutically acceptable solvate thereof, is astereoselective procedure and that the compound of formula (I) is asingle stereoisomer. Favourably a compound of formula (I) is present inadmixture with less than 50% w/w of its racemic isomer, that is when itis greater than 50% optically pure, suitably 80-100% and preferably90-100% pure, such as 90-95%, most preferably 95-100%, for example 95%,96%, 97%, 98%, 99% or 99.9% optically pure.

Preferably the compound of formula (I) or a pharmaceutically acceptablesalt thereof and/or a pharmaceutically acceptable solvate thereof, is inoptically pure form.

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography.

When the agonist of PPARα and PPARγ is provided by the combination of aPPARα agonist compound and a PPARγ agonist compound, a suitable PPARγagonist compound is selected from EP 0306228 and WO94/05659, thecontents of which are incorporated herein by reference.

Suitable, favoured and preferred PPARγ agonists are those suitable,favoured and preferred compounds disclosed in EP 0306228 and WO94/05659.

A most preferred PPARγ agonist from EP 0306228 and WO94/05659 is5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dioneor a tautomeric form thereof and/or a pharmaceutically acceptable saltthereof, especially a maleic acid salt thereof, and/or apharmaceutically acceptable solvate thereof.

Additional PPARγ agonists include the thiazolidinediones disclosed inEuropean Patent Applications, Publication Numbers:0008203, 0139421,0032128, 0428312, 0489663, 0155845, 0257781, 0208420, 0177353, 0319189,0332331, 0332332, 0528734 and 0508740; and from International PatentApplication, Publication Numbers 92/18501, 93/02079, 93/22445 and fromU.S. Pat. No. 5,104,888; the contents of these publications are alsoincluded herein by reference.

When the agonist of PPARα and PPARγ is provided by the combination of aPPARα agonist compound and a PPARγ agonist compound, suitable PPARαagonist are the fibrates such as clofibrate, ciprofibrate, Wy 14643 andBR-931 (Lalwani et al, Biochemical and Biophysical Research commun.,Vol. 116, 388-393, 1983); the contents of these publications areincluded herein by reference. The said fibrates are known compoundsprepared using known methodology or analogous methodology to that use toprepare known, analogous compounds, for example the method of d'Atri etal J. Med. Chem., Vol 27, 1621-1629, 1984 is generally applicable toeach of the mentioned fibrates.

Also specifically included in the method of the invention are thespecific examples disclosed in the above mentioned publicationsincluding the patent applications.

The active compounds disclosed in the above mentioned published patentpublications, including the specific examples disclosed therein, areconveniently prepared according to the methods disclosed in the saidpatent publications: For example a PPARγ agonist selected from EP0306228 or WO94/05659 can be prepared using the processes described inEP 0306228 and WO94/05659.

When used herein `Syndrome X` includes pre-diabetic insulin resistancesyndrome and the resulting complications thereof, insulin resistance,non-insulin dependent diabetes, dyslipidaemia, hyperglycaemia, obesityand the complications associated with diabetes; the methods andtreatments mentioned herein include the above unless specifically statedotherwise.

For the avoidance of doubt, the methods and treatments of this inventionalso encompass the treatment and/or prophylaxis of any one of or anycombination of the following list: pre-diabetic insulin resistancesyndrome, the resulting complications thereof, insulin resistance,non-insulin dependent diabetes, dyslipidaemia, hyperglycaemia, obesityand the complications associated with diabetes.

The complications associated with diabetes include cardiovasculardisease, especially atherosclerosis, retinopathy, neuropathy and renaldisease including diabetic nephropathy, glomerulonephritis, glomerularsclerosis, nephrotic syndrome, hypertensive nephrosclerosis and endstage renal disease.

When used herein the term `PPARα agonist` relates to an agonist of theperoxisomal proliferator-activated receptor, suitably the humanreceptor, of the alpha subtype.

When used herein the term `PPARγ agonist` relates to an agonist of theperoxisomal proliferator-activated receptor, suitably the humanreceptor, of the gamma subtype.

PPARα and γ agonist activity may be assessed by use of the methodology,or similar methodology, to that disclosed by Lehmann et al: Journal ofBiological Chem., 270, 12953-12956 (1995).

In one aspect PPARα agonist compounds are those which stimulate a PPARalpha chimeric receptor consisting of the PPAR alpha ligand binding sitelinked to a suitable reporter gene construct such as luciferase orchloramphenicol acetyltransferase (CAT). This activity can be identifiedby using the methods outlined by Lehmann et al., ibid.

In one aspect PPARγ agonist compounds are those which stimulate a PPARγchimeric receptor containing the PPAR gamma ligand binding site linkedto a suitable reporter gene construct such as luciferase orchloramphenicol acetyltransferase (CAT). This activity can be identifiedby using the methods of Lehmann et al. J Biol. Chem., ibid.

Agonists may be proteins or non-proteins.

Suitable agonists are small molecular weight, non-protein compounds.

Also included in the present invention is a method for detecting acompound having both PPARα and PPARγ agonist activity.

Suitable methods of detection include determining:

(a) PPARα agonist activity by detecting stimulation of a PPAR alphachimeric receptor consisting of the PPAR alpha ligand binding sitelinked to a suitable reporter gene construct; and

(b) PPARγ agonist activity by detecting stimulation of a PPARγ chimericreceptor containing the PPAR gamma ligand binding site linked to asuitable reporter gene construct such as luciferase or chloramphenicolacetyltransferase (CAT).

A suitable PPARα chimeric receptor comprises the amino acids of theligand binding domain, for example amino acids 281-468, of the humanPPARα, fused to amino acids 1-147 (DNA binding domain) of the gal 4yeast transcription factor.

A suitable PPARγ chimeric receptor comprises the amino acids of theligand binding domain, for example amino acids 173-476, of the humanPPARγ receptor fused to amino acids 1-147 (DNA binding domain) of thegal 4 yeast transcription factor.

A suitable reporter gene construct is a luciferase or chloramphenicolacetyltransferase (CAT).

A suitable luciferase reporter gene construct contains gal 4 DNA bindingelements in HEK-293 cells.

Suitable methodology for the said method of detection is as describedabove.

The present invention also provides an agonist of PPARα and PPARγ, foruse in the treatment and/or prophylaxis of Syndrome X.

A particular treatment is the prophylaxis of hyperglycaemia.

A particular treatment is the treatment of pre-diabetic insulinresistance syndrome and the resulting complications thereof.

Also included is the treatment and/or prophylaxis of hyperglycaemiaand/or pre-diabetic insulin resistance syndrome and the resultingcomplications thereof; providing the said treatment does not includeadministration of:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid, for the treatment and/or prophylaxis of: hyperglycaemia,especially in Type II diabetes, hyperlipidaemia, hypertension,cardiovascular disease, especially atherosclerosis and of renal disease,especially renal disease associated with the development of Type IIdiabetes including diabetic nephropathy, glomerulonephritis, glomerularsclerosis, nephrotic syndrome, hypertensive nephrosclerosis and endstage renal disease and for the prevention, reversal, stabilisation orretardation of the progression of microalbuminuria to albuminuria.

The present invention also provides an agonist of PPARα and PPARγ, foruse in the manufacture of a medicament the treatment and/or prophylaxisof Syndrome X, and in particular for the treatment and/or prophylaxis ofhyperglycaemia and/or pre-diabetic insulin resistance syndrome and theresulting complications thereof; providing the said treatment does notinclude administration of:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid, for the treatment and/or prophylaxis of: hyperglycaemia,especially in Type II diabetes, hyperlipidaemia, hypertension,cardiovascular disease, especially atherosclerosis and of renal disease,especially renal disease associated with the development of Type IIdiabetes including diabetic nephropathy, glomerulonephritis, glomerularsclerosis, nephrotic syndrome, hypertensive nephrosclerosis and endstage renal disease and for the prevention, reversal, stabilisation orretardation of the progression of microalbuminuria to albuminuria.

This is considered to be the first indication of a compound havingagonist activity at both PPARα and PPARγ. Other compounds having thisdual activity would also be of particular use for the treatment and/orprophylaxis of Syndrome X, including hyperglycaemia and pre-diabeticinsulin resistance syndrome and the resulting complications thereof.

Accordingly, in a further aspect the present invention also provides acompound having agonist activity at both PPARα and PPARγ.

Suitable compounds are unique, in that they are not known to haveagonist activity at both PPARα and PPARγ or they are novel compounds perse.

In one aspect the said compound having agonist activity at both PPARαand PPARγ does not include:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid.

The invention also provides a compound for use as an agonist of bothPPARα and PPARγ.

The invention further provides the a compound having agonist activity atboth PPARα and PPARγ for use as an active therapeutic substance;suitably providing that the compound does not include:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid.

In the above mentioned treatments the active compound is administeredper se or, preferably, as a pharmaceutical composition also comprising apharmaceutically acceptable carrier.

Accordingly, the present invention also provides a pharmaceuticalcomposition comprising an agonist of PPARα and PPARγ and apharmaceutically acceptable carrier therefor; suitably providing thesaid agonist does not include:

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid; or

(S)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid.

In addition there is provided a method for treating conditions caused bya requirement for an agonist of both PPARα and PPARγ in a human ornon-human mammal, which method comprises the administration of aneffective, pharmaceutically acceptable, non-toxic amount of an agonistof both PPARα and PPARγ.

As used herein the term `pharmaceutically acceptable` embracescompounds, compositions and ingredients for both human and veterinaryuse: for example the term `pharmaceutically acceptable salt` embraces aveterinarily acceptable salt.

The composition may, if desired, be in the form of a pack accompanied bywritten or printed instructions for use.

Usually the pharmaceutical compositions of the present invention will beadapted for oral administration, although compositions foradministration by other routes, such as by injection and percutaneousabsorption are also envisaged.

Particularly suitable compositions for oral administration are unitdosage forms such as tablets and capsules. Other fixed unit dosageforms, such as powders presented in sachets, may also be used.

In accordance with conventional pharmaceutical practice the carrier maycomprise a diluent, filler, disintegrant, wetting agent, lubricant,colourant, flavourant or other conventional adjuvant.

Typical carriers include, for example, microcrystalline cellulose,starch, sodium starch glycollate, polyvinylpyrrolidone,polyvinylpolypyrrolidone, magnesium stearate, sodium lauryl sulphate orsucrose.

Most suitably the composition will be formulated in unit dose form. Suchunit dose will normally contain an amount of the active ingredient inthe range of from 0.1 to 1000 mg, more usually 0.1 to 500 mg, and moreespecially 0.1 to 250 mg.

Conveniently, the active ingredient may be administered as apharmaceutical composition hereinbefore defined, and this forms aparticular aspect of the present invention.

In the above mentioned treatments the active compounds are suitablytaken in doses such as those described above, one to six times a day ina manner such that the total daily dose for a 70 kg adult will generallybe in the range of from 0.1 to 6000 mg, and more usually about 1 to 1500mg, generally about 0.5 to 10 mg. That is in the range of from1.429×10⁻³ to 85.714 mg/kg/day, more usually about 1.429×10⁻² to 21.429mg/kg/day, generally about 7.143×10⁻³ to 0.1429 mg/kg/day.

No adverse toxicological effects are expected when a compound isadministered in accordance with the above mentioned invention.

The activity of the present compounds can be demonstrated using themethods disclosed below. The following Preparations illustrate thepreparation of the compounds of formula (I).

PREPARATION OF COMPOUNDS OF FORMULA (I) Preparation 1:(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid ##STR12##

A solution of[2S,N(1S)]-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)-N-(2-hydroxy-1-phenylethyl)propanamide(1.846 g) in a mixture of 1M sulphuric acid (45 mL) and dioxan/water(1:1, 150 mL) was heated at 90° C. for 56 hours and then the pH of themixture was adjusted to pH 3 by addition of aqueous sodium hydrogencarbonate. The mixture was extracted with ethyl acetate and the organicextracts washed with water, brine, dried (MgSO₄) and evaporated to givean oil. Purification by chromatography on silica gel using a gradient of1-5% methanol in dichloromethane as eluent gave a foam of 88% e.e. (byHPLC). The product was reacted with (S)-α-methylbenzylamine in acetone,and the resulting salt recrystallised several times from ethylacetate-hexane before being dissolved in water, acidified with dilutehydrochloric acid and extracted with ethyl acetate which was dried withMgSO₄. Evaporation of the ethyl acetate solution affordedenantiomerically enriched title compound; [α]_(D) ²⁵ -28° (c=0.625,CHCl₃); e.e 94% (by HPLC); [Found M⁺ 414.1791. C₂₂ H₂₆ N₂ O₆ requires M⁺414.1791]; ¹ H NMR spectrum identical with that described in Example 5.

Preparation 2:(S)-3-(4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid by Hydrolysis of Amide ##STR13##

[2S,N(1S)]-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)-N-(2-hydroxy-1-phenylethyl)propanamide(from Procedure 3) was hydrolysed by an analogous procedure to thatdescribed in Preparation 1. Purification by chromatography on silica gelusing a gradient of 0-5% methanol in dichloromethane as eluent gave thetitle compound, mp 116-7° C., after trituration with diethylether-hexane; [α]_(D) ²⁵ -24.6° (c=0.24, CHCl₃); e.e. 95% (by HPLC).[Found C, 57.9; H, 4.7; N, 6.8%; M⁺ 438.1403. C₂₁ H₂₁ F₃ N₂ O₅ requiresC, 57.5; H, 4.8; N, 6.4%; M⁺ 438.1403]; δ_(H) (DMSO-d₆) 2.96 (2H,m),3.22 (3H,s), 3.88 (2H,m), 3.95-4.18 (2H,m), 4.27 (3H,m), 6.8-7.37 (8H,m)and 12.9 (1H,br s, exchanges with D₂ O).

Preparation 3:(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl-2-(2,2,2-trifluoroethoxy)propanoicAcid, by Direct Hydrolysis of the Imide ##STR14##

Aqueous sodium hydroxide solution (2.5M, 65 mL, 0.163 mol, 2.3 eq) wasadded to a stirred solution of[3(2S),4S]-3-[3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-one(from Procedure 10)(42.5 g, 0.071 mol) in THF (500 mL) and water (125mL). The mixture was stirred for 20 minutes, the reaction was dilutedwith water (1 L) and extracted with dichloromethane (3×700 mL). Thesedichloromethane solutions were evaporated and the residue purified bychromatography on silica gel using 5% methanol in dichloromethane aseluent to afford (S)-4-benzyloxazolidin-2-one. The original aqueoussolution was acidified to pH 3.5 with dilute hydrochloric acid andre-extracted with dichloromethane (3×700 mL). The dichloromethanesolutions from the acid extraction were dried (MgSO₄) and evaporated togive a solid. This was recrystallised from dichloromethane-diethyl etherto afford the title compound, mp 119.5-120.5° C. [α]_(D) ²⁵ =-31°(c=2.50, CHCl₃); e.e. 99.6% (by HPLC); [Found C, 57.7; H, 4.7; N, 6.25%;M⁺ (EI) 438.1412. C₂₁ H₂₁ F₃ N₂ O₅ requires C, 57.5; H, 4.8; N, 6.4%; M⁺438.1403]; δ_(H) (CDCl₃) 3.05 (1H, dd), 3.13 (1H, dd), 3.31 (3H, s),3.72 (1H, m), 3.89 (2H, m), 4.04-4.14 (3H, m), 4.21 (1H, dd), 6.78 (2H,d), 7.03-7.40 (6H, m) and 11.20 (1H, br, exchanges with D₂ O); δ_(F)(DMSO-d₆)=-72.7 (3F, t, ³ J_(HF) 9.3 Hz, CF₃).

Preparation 4:(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl-2-(2,2,2-trifluoroethoxy)propanoicAcid by Hydrolysis of Methyl Ester ##STR15##

A mixture of (S)-methyl3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoate(1.256 g, 2.8×10⁻³ mol), aqueous hydrochloric acid (2.0M, 50 mL) anddioxan (50 mL) was heated at reflux for 7 hours, cooled and concentratedin vacuo. The residue was suspended in brine (200 mL) and extracted withethyl acetate (3×300 mL). The combined ethyl acetate solutions weredried (MgSO₄) and evaporated to afford a waxy solid. This solid wastriturated with hexane, filtered and dried under vacuum at 65° C. toafford the desired product, mp 113-5° C. [α]_(D) ²⁵ =-32° (c=1.02,CHCl₃); e.e. 99.4% (by HPLC); [Found C, 57.25; H, 4.8; N, 6.3%. C₂₁ H₂₁F₃ N₂ O₅ requires C, 57.5; H, 4.8; N, 6.4%]. The ¹ H NMR spectrum ofthis material was identical to that produced in Example 3.

Preparation 5:(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl-2-(2-methoxyethoxy)propanoicAcid ##STR16##

(S)-Methyl3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoatewas hydrolysed in a manner analogous to that described for Example 4.The crude reaction mixture was chromatographed on silica gel using 5%methanol in dichloromethane as eluent to afford the title compound, agum. [α]_(D) ²⁵ =-27° (c=0.73, CHCl₃); e.e. 99.8% (by HPLC); (Found M⁺(EI)414.1779. C₂₂ H₂₆ N₂ O₆ requires M⁺ 414.1791]; δ_(H) (CDCl₃) 2.90(1H, dd), 3.15 (1H, dd), 3.33 (3H, s), 3.37 (3H, s), 3.40-3.70 (4H, m),3.93 (2H, t), 4.05 (1H, dd), 4.21 (2H, t), 6.81 (2H, d) and 6.95-7.40(6H,m).

Preparation 6:(±)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy]propanoicacid ##STR17##

A mixture of methyl3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoate(1.08 g, Int. Patent Appl., Publication No. WO 9401420) and sodiumhydroxide (253 mg) in methanol:water (1:1, 10 mL) was heated underreflux for 2 hours. After evaporation of the resultant mixture in vacuo,the residue was diluted with water, acidified to pH 5 with 2Mhydrochloric acid and then extracted with ethyl acetate. Washing of theethyl acetate extracts with water and drying (MgSO₄) and evaporationgave the title compound as an oil which crystallised on trituration withdiethyl ether/hexane. [Found C, 63.8; H, 6.5; N, 7.0%; M⁺ 414.1791. C₂₂H₂₆ N₂ O₆ requires C, 63.8; H, 6.3; N, 6.8%; M⁺ 414.1791]; δ_(H) (CDCl₃)2.91 (1H,dd), 3.15 (1H,dd), 3.34 (3H,s), 3.38 (3H,s), 3.41-3.69 (4H,m),3.93 (2H,t), 4.05 (1H,dd), 4.21 (2H,t), 6.80 (2H,d) and 6.83-7.38 (6Hm).

PROCEDURE 2 Preparation 7:(±)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoylchloride ##STR18##

Oxalyl chloride (92 mg) was added to(±)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoicacid (100 mg) in dichloromethane (2 mL). The mixture was stirred at roomtemperature for 16 hours and evaporated to dryness to give the titlecompound as a gum which was used without further purification.

Preparation 8:[2S,N(1S)]-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)-N-(2-hydroxy-1-phenylethyl)propanamide##STR19##

(+)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoylchloride was dissolved in dichloromethane (2 mL) and a mixture of(S)-2-phenylglycinol (33 mg) and dry triethylamnine (37 mg) indichloromethane (1 mL) added. After stirring for 5 minutes water wasadded and the mixture extracted with dichloromethane. The organicextracts were washed with water, brine, dried (MgSO₄) and evaporated.The residue was chromatographed on silica gel using a gradient of 10-50%acetone in hexane as eluent to afford firstly[2R,N(1S)]-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)-N-(2-hydroxy-1-phenylethyl)propanamidefollowed by the desired [2S,N(1S)]-propanamide title compound as a foam.[α]_(D) ²⁵ -33° (c=1.1, CHCl₃); 92.6% d.e. (by HPLC); [Found M⁺533.2526. C₃₀ H₃₅ N₃ O₅ requires M⁺ 533.2526]; δ_(H) (CDCl₃) 2.81(1H,dd), 3.07 (1H,dd), 3.35 (3H,s), 3.36 (3H,s), 3.48-3.58 (2H,m),3.52-3.62 (2H,m), 3.71 (1H,dd), 3.82 (1H,dd), 3.94 (1H,dd), 3.93 (2H,t),4.22 (3H,t), 5.05 (1H,dt), 6.75-7.35 (13H,complex), 7.54 (1H,br,exchanges with D₂ O).

PROCEDURE 4 Preparation 9:(±)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid ##STR20##

Methyl3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoate(Int. Patent Appl., Publication No. WO 9401420) was hydrolysed by ananalogous procedure to that described in Preparation 5 to give the titlecompound as a solid, mp 116-117° C.; [Found C, 57.4; H, 4.9; N, 6.4%.C₂₁ H₂₁ F₃ N₂ ₅ requires C, 57.5; H, 4.8; N, 6.4%); δ_(H) (CDCl₃)3.03-3.17 (2H,m), 3.29 (3H,s), 3.73-3.83 (1H,m), 3.85 (2H,m), 4.02(2H,m), 4.04-4.30 (2H,m) and 6.74-7.40 (8H m).

PROCEDURE 5 Preparation 10:(±)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoylchloride ##STR21##

Oxalyl chloride (1.1 mL) was added to a solution of(±)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoicacid (1.72 g) in dry benzene (30 mL). The mixture was heated at refluxfor 2 hours, cooled and evaporated to dryness to give the title compoundas a gum which was used without further purification.

PROCEDURE 6 Preparation 11:[2S,N(1S)]-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)-N-(2-hydroxy-1-phenylethyl)propanamide##STR22##

(±)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoylchloride was reacted with (S)-2-phenylglycinol by an analogous procedureto that described in Procedure 3. Chromatography on silica gel using agradient of 10-70% ethyl acetate in hexane as eluent afforded firstly[2R,N(1S)]-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)-N-(2-hydroxy-1-phenylethyl)propanamidefollowed by the desired [2S,N(1S)]-propanamide title compound as a foam;[α]_(D) ²⁵ +14° (c=0.5, MeOH); 99% d.e. (by HPLC); [Found M⁺ 557.2136.C₂₉ H₃₀ F₃ N₃ O₅ requires M⁺ 557.2138]; δ_(H) (CDCl₃) 2.35 (1H,br,exchanges with D₂ O), 2.91 (1H,dd), 3.13 (1H,dd), 3.36 (3H,s), 3.70-3.87(2H,m), 3.84 (2H,d), 3.95 (2H,t), 4.12 (1H,dd),4.22 (2H,t), 5.01 (1H,m),6.75 (2H,d), 6.97 (1H,br s, exchanges with D₂ O) and 7.01-7.36(11H,complex).

Preparation 12: (2,2,2-Trifluoroethoxy)ethanoyl Chloride ##STR23##

A solution of oxalyl chloride (20 mL, 0.23 mol, 1.15 eq) in drydichloromethane (50 mL) was added dropwise at room temperature, withstirring, to a solution of (2,2,2-trifluoroethoxy)ethanoic acid (int.Patent Appl., Publication No. WO 87/07270, 31.6 g, 0.2 mol) andN,N-dimethylformamide (5 drops) in dry dichloromethane (400 mL). Themixture was stirred for an additional hour, then heated under reflux for2 hours, cooled and the bulk of the solvent removed by distillation (bp40-45° C./760 mm Hg). The residue was transferred to a Claisendistillation flask and the remaining solvent and oxalyl chloride removedby distillation (bp 45-60° C./760 mm Hg). Vacuum distillation of theresidue then afforded the product, bp 50-55°/25-32 mm Hg. δ_(H) (CDCl₃)4.00 (2H, q, ³ J_(HF) 8.3) and 4.57 (2H, s).

PROCEDURE 8 Preparation 13:(4S)-4-Benzyl-3-[2-(2,2,2-trifluoroethoxy)ethanoyl]oxazolidin-2-one##STR24##

(4S)-4-Benzyloxazolidine-2-one (5.21 g, 0.029 mol) was dissolved in dryTHF (60 mL) and cooled to -70° C. under argon. n-Butyllithium (18.4 mL,1.6 M solution in hexane, 1.1 eq) was added over 10 minutes and theresulting mixture stirred at -70° C. for 20 minutes. A solution of(2,2,2-trifluoroethoxy)ethanoyl chloride (5.19 g, 1 eq) in dry TUF (60mL) was added over 10 minutes, the mixture stirred at -70° C. for afurther 30 minutes then allowed to warm to room temperature overnight.The reaction was quenched by addition of brine (20 mL) and concentratedin vacuo. The residue was diluted with brine (300 mL) and extracted withethyl acetate (3×300 mL). The combined organic extracts were dried(MgSO₄), evaporated and the residue chromatographed on silica gel withdichloromethane as eluent to give the product as an oil. [α]_(D) ²⁵=+48° (c=2.55, CHCl₃); e.e. 100% (by HPLC); [Found (CI, Ammonia) MH⁺318.0934. C₁₄ H₁₄ NO₄ F₃ requires MH⁺ 318.0953]; δ_(H) (CDCl₃) 2.82 (1H,dd), 3.34 (1H, dd), 4.02 (2H, q, ³ J_(HF) 8.6), 4.30 (2H, m), 4.69 (1H,m), 4.84 (2H, s) and 7.15-7.40 (5H, m); δ_(F) (CDCl₃)=-74.8 (3F, t, ³J_(HF) 8.6, CF₃).

PROCEDURE 9 Preparation 14:[3(2S,3R),4S]-3-[3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-3-hydroxy-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-one##STR25##

(4S)-4-Benzyl-3-[2-(2,2,2-trifluoroethoxy)ethanoyl]oxazolidin-2-one(31.7 g, 0.1 mol) was dissolved in dry dichloromethane (300 mL) underargon and cooled to -78° C. (internal temperature of solution), usingliquid nitrogen/acetone as the cooling medium. Triethylamine (16.72 mL,1.2 eq) was added, followed by the slow addition, over approximately 10minutes, of di-n-butylboron triflate (Aldrich Chemical Company, 1.0Msolution in dichloromethane, 110 mL, 1.1 eq) such that the reactiontemperature was maintained below -70° C. The mixture was stirred at -78°C. for 50 minutes, then the cooling bath was replaced with an ice bathand the mixture stirred at 0° C. for an additional 50 minutes beforebeing recooled to -78° C. A solution of4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]benzaldehyde (29.6 g, 1.0eq) in dry dichloromethane (220 mL), precooled to -50° C., was addedover ca. 12 minutes, such that the reaction temperature was maintainedbelow -70° C. The resulting mixture was stirred at -78° C. for 30minutes, then warmed from -78° C. to 0° C. over 60 minutes along alinear gradient (warming rate ˜1.3° C. min⁻¹) and stirred at 0° C. for afurther 75 minutes. The reaction mixture was poured into a quenchingsolution of methanol (500 mL), pH 7 phosphate buffer (250 mL) andhydrogen peroxide (27.5% w/v, 110 mL) and stirred vigourously for 30minutes. Water (4 L) was added, the layers were separated and theaqueous layer was extracted with dichloromethane (3×1 L). Thedichloromethane solutions were recombined with the originaldichloromethane layer from the reaction mixture and this organicsolution was then washed with water (2 L) and brine (2 L), dried (MgSO₄)and evaporated to afford a foam. ¹ H NMR of this crude reaction mixturesuggested a mixture of the desired aldol product (3 diastereoisomers,comprising 95% major diastereoisomer) and starting materials. The crudemixture was chromatographed on silica gel using a gradient elutioncomprising 15% ethyl acetate in dichloromethane initially (until thedesired product began to elute) and rising to 50% ethyl acetate indichloromethane to complete the elution of the desired product.Unreacted imide and aldehyde were recovered from the early fractions,followed by a quantity of impure product and then the title compound(comprising 2 diastereoisomers, ratio 97.8:2.2 by NMR). [α]_(D) ²⁵ =+45°(c=2.82, CHCl₃). [Found (EI) M⁺ 613.2042. C₃₁ H₃₀ F₃ N₃ O₇ requires M⁺613.2036]; δ_(H) (CDCl₃, only major diastereoisomer is recorded) 2.75(1H, dd), 2.90 (1H, d, exchanges with D₂ O), 3.25 (1H, dd), 3.34 (3H,s), 3.80-4.00 (5H, m), 4.07 (1H, dd), 4.24 (2H, t), 4.45 (1H, m), 4.99(1H, apparent t), 5.48 (1H, d), 6.85 (2H, d) and 6.95-7.40 (11H, m);δ_(F) (CDCl₃)=-74.7 (3F, t, ³ J_(HF) 8.5, CF₃). The minordiastereoisomer in the purified product was identified as the[3(2S,3S),4S]-diastereoisomer.

PROCEDURE 10 Preparation 15:[3(2S),4S]-3-[3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-oneby Dehydroxylation ##STR26##

Triethylsilane (120 mL, 0.75 mol) was added over 5 minutes to a stirred,ice cooled solution of[3(2S,3R),4S]-3-[3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-3-hydroxy-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-one(46.23 g, 7.5×10⁻² mol) in trifluoroacetic acid (650 mL). The mixturewas stirred at 0° C. for 1 hour, then at room temperature for a further60 hours. The bulk of the solvent and residual triethylsilane wasremoved by rotary evaporation, firstly at 40 mm Hg and finally at ˜5 mmHg. The residue was dissolved in dichloromethane (800 mL) and water (800mL), then stirred vigorously during the cautious addition of solidsodium bicarbonate (˜29 g) (frothing !) until the pH of the aqueouslayer was pH 7. The layers were separated and the aqueous layer wasextracted with dichloromethane (800 mL). The combined dichloromethanelayers were washed with water (600 mL), dried (MgSO₄) and evaporated.The residue was triturated with hot hexane and the resulting solidcollected by filtration. Recrystallisation from diethyl ether-hexaneafforded the title compound, mp 107-109° C., a single diastereoisomer by¹ H NMR spectroscopy. [α]_(D) ²⁵ =+38° (c=1.51, CHCl₃); [Found C, 62.1;H, 4.9; N, 7.2%; M⁺ (EI) 597.2089. C₃₁ H₃₀ N₃ O₆ F₃ requires C, 62.3; H,5.1; N, 7.0%; M⁺ 597.2087]; δ_(H) (CDCl₃) 2.82 (1H, dd), 2.96 (1H, dd),3.04 (1H, dd), 3.32 (1H, dd), 3.34 (3H, s), 3.70 (1H, m), 3.88 (1H, m),3.94 (2H, t), 4.12 (1H, m), 4.18 (1H, m), 4.25 (2H, t), 4.57 (1H, m),5.34 (1H, dd), 6.82 (2H, d) and 7.00-7.35 (11H, m); δ_(F) (CDCl₃)=-74.8(3F, t, ³ J_(HF) 8.6, CF₃).

PROCEDURE 11 Preparation 16:[3(2S),4S]-3-[3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-onevia Diastereoisomer Separation ##STR27##

(S)-4-Benzyloxazolidin-2-one (0.291 g, 1.64×10⁻³ mol) was dissolved indry THF (10 mL) and the resulting solution cooled to -70° C. underargon. n-Butyl lithium (1.6M in hexane, 1.03 mL, 1.64×10⁻³ mol) wasadded and the mixture was stirred at -70° C. for 10 minutes prior to theaddition of a solution of(±)-3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoylchloride (prepared from 0.36 g of the acid by Procedure 5, above) in dryTHF (15 mL). The reaction was stirred and allowed to warm to roomtemperature overnight before being diluted with water (200 mL) andextracted with ethyl acetate (2×200 mL). The combined ethyl acetatelayers were washed with water (200 mL) and brine (200 mL), dried (MgSO₄)and evaporated to give a brown gum. This was chromatographed on silicagel using a gradient of 35% to 50% ethyl acetate in hexane as eluent toafford firstly the (R,S)-diastereoisomer, followed by the titlecompound, a foam. This material was spectroscopically identical withthat prepared by the aldol route (Procedure 10).

PROCEDURE 12 Preparation 17: (S)-Methyl3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoate##STR28##

A solution of sodium methoxide [prepared from sodium hydride (60%dispersion in mineral oil, 138 mg, 3.41×10⁻³ mol) dissolved in drymethanol (3.5 mL)] was added to an ice cooled and stirred suspension of[3(2S),4S]-3-[3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2,2,2-trifluoroethoxy)propanoyl]-4-benzyloxazolidin-2-one(1.879 g, 3.1×10⁻³ mol) in dry methanol (100 mL). The mixture wasstirred at 0° C. for a total of 20 minutes, then the reaction wasquenched by the addition of dilute aqueous hydrochloric acid (2.0M, 1.75mL) and concentrated in vacuo. The residue was suspended in water (100mL), extracted with ethyl acetate (3×200 mL) and the combined ethylacetate solutions washed with brine (500 mL), dried (MgSO₄) andevaporated. The resulting gum was chromatographed on silica gel using 4%ethyl acetate in dichloromethane as eluent to afford the product as aclear gum. [α]_(D) ²⁵ =-17° (c=1.24, CHCl₃); [Found (EI) M⁺ 452.1561.C₂₂ H₂₃ N₂ O₅ F₃ requires M⁺ 452.1559]; e.e. 100% (by HPLC); δ_(H)(CDCl₃) 3.02 (2H, m), 3.34 (3H, s), 3.65 (1H, m), 3.72 (3H, s), 3.94(2H, t), 4.00 (1H, m), 4.13 (1H, dd), 4.24 (2H, t), 6.80 (2H, d) and6.96-7.40 (6H, m).

PROCEDURE 13 Preparation 18:(4S)-4-Benzyl-3-[2-(2-methoxyethoxy)ethanoyl]oxazolidin-2-one ##STR29##

The title compound was prepared from 2-(2-methoxyethoxy)ethanoylchloride by a method analogous to that described in Procedure 8.Chromatography on silica gel using a gradient of 70-80% diethyl ether inhexane as eluent afforded the product as a gum. [α]_(D) ²⁵ =+54°(c=2.70, CHCl₃); [Found (EI) M⁺ 293.1263. C₁₅ H₁₉ NO₅ requires M⁺293.1264]; δ_(H) (CDCl₃) 2.81 (1H, dd), 3.33 (1H, dd), 3.41 (3H, s),3.63 (2H, t), 3.78 (2H, t), 4.25 (2H, m), 4.70 (1H, m), 4.74 (1H, d),4.76 (1H, d) and 7.10-7.40 (5H, m).

PROCEDURE 14 Preparation 19:[3(2S,3R),4S]-3-[3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-3-hydroxy-2-(2-methoxyethoxy)propanoyl]-4-benzyloxazolidin-2-one##STR30##

The title compound was prepared from(4S)-4-benzyl-3-[2-(2-methoxyethoxy)ethanoyl]oxazolidin-2-one by amethod analogous to that described in Procedure 9. The crude reactionmixture was chromatographed on silica gel using a gradient of 15-40%ethyl acetate in dichloromethane to afford the product as a gum(comprising 2 diastereoisomers, ratio >99:1 by ¹ H NMR). [α]_(D) ²⁵=+49° (c=1.14, CHCl₃). [Found (FAB, NOBA/Na) MH⁺ 590.2472. C₃₂ H₃₅ N₃ O₈requires MH⁺ 590.2502]; δ_(H) (CDCl₃, only major diastereoisomer isrecorded) 2.71 (1H, dd), 3.25 (1H, dd), 3.31 (3H, s), 3.35 (3H, s), 3.56(2H, m), 3.72 (2H, m), 3.78 (1H, d, exchanges with D₂ O), 3.85-4.00 (4H,m), 4.22 (2H, t), 4.31 (1H, m), 4.89 (1H, dd), 5.42 (1H, d), 6.83 (2H,d) and 6.95-7.40 (11H, m); The minor diastereoisomer in the purifiedproduct was identified as the [3(2S,3S),4S]-diastereoisomer.

PROCEDURE 15 Preparation 20:[3(2S),4S]-3-[3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoyl]-4-benzyloxazolidin-2-one##STR31##

[3(2S,3R),4S]-3-[3-[4-[2-IN-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-3-hydroxy-2-(2-methoxyethoxy)propanoyl]-4-benzyloxazolidin-2-one(0.561 g) was reacted with triethylsilane for 6.25 hrs in a mannersimilar to that described for Procedure 10. The reaction mixture wasdiluted with water (200 mL) and dichloromethane (200 mL) and solidsodium bicarbonate was added cautiously until the aqueous layer showedpH 6.5. The layers were separated, the aqueous layer was extracted withdichloromethane (2×300 mL) and the combined dichloromethane solutionswere washed with brine (400 mL), dried (MgSO₄) and evaporated. Theresidue was chromatographed on silica gel using 35% ethyl acetate indichloromethane as eluent to afford the title compound, a gum, as asingle diastereoisomer by ¹ H NMR. [α]_(D) ²⁵ =+45° (c=1.39, CHCl₃);[Found M⁺ (EI) 573.2473. C₃₂ H₃₅ N₃ O₇ requires M⁺ 573.2475]; δ_(H)(CDCl₃) 2.76 (1H, dd), 2.94 (2H, m), 3.30 (3H, s), 3.33 (4H, m),3.40-3.70 (4H, m), 3.93 (2H, t), 4.00 (1H, dd), 4.12 (1H, dd), 4.22 (2H,t), 4.52 (1H, m), 5.31 (1H, dd), 6.79 (2H, d) and 6.90-7.40 (11H, m).

PROCEDURE 16 Preparation 21: (S)-Methyl3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(2-methoxyethoxy)propanoate##STR32##

[3(2S),4S]-3-[3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]phenyl]-2-(Z2-methoxyethoxy)propanoyl]-4-benzyloxazolidin-2-onewas reacted with sodium methoxide in a manner analogous to thatdescribed in Procedure 12. The crude reaction mixture waschromatographed on silica gel using 20% isohexane in diethyl ether aseluent to afford the title compound, a gum. [α]_(D) ²⁵ =-12° (c=1.26,CHCl₃); [Found (EI) M⁺ 428.1974. C₂₃ H₂₈ N₂ O₆ requires M⁺ 428.1948];e.e. >99.8% (by HPLC); δ_(H) (CDCl₃) 2.95 (2H, m), 3.29 (3H, s), 3.34(3H, s), 3.35 (3H, m), 3.69 (4H, m), 3.93 (2H, t), 4.05 (1H, dd), 4.23(2H, t) and 6.75-7.40 (8H, m).

Demonstration of Efficacy of Compounds

1) Determination of hPPARα and hPPAR-γ Agonist Activity

Compound agonist effects at human PPARα and PPARγ were assessed using atransactivation reporter gene assay, based on that described by Lehmanet al. (1995) J Biol Chem 270, 12953-12956.

Results: Efficacy of(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]2-(2,2,2-trifluoroethoxy)propanoicacid at hPPARα and hPPARγ

EC₅₀ for activation of human PPARα is=2500 nM

EC₅₀ for activation of human PPARγ is=70 nM

2) Determination of Compound Efficacy on Blood Glucose and Plasma Lipidsin the C57 Bl/KsJ db/db Mouse

The genetically diabetic C57 Bl/KsJ db/db mouse displays a severe formof non insulin dependent diabetes mellitus in that it develops aprofound hyperglycaemia at about 8 weeks of age. This is paralleled byglycosuria and polyuria with a compensatory increase in water intake.Circulating serum triglycerides and free fatty acids are also elevated.

Compounds are administered by dietary admixture (powdered RM3 diet) for14 days and blood glucose measured in samples taken from the tail veinof conscious non-fasted mice at appropriate intervals during thetreatment period. After the 14 day treatment period, mice are killed bycervical dislocation and blood obtained from the severed jugular veins.Triglyceride and non-esterified fatty acids are measured in samples ofserum obtained by centrifugation.

Efficacy of(S)-3-[4-[2-[N-(2-Benzoxazolyl)-N-methylamino]ethoxy]phenyl]2-(2,2,2-trifluoroethoxy)propanoicacid on Blood Glucose in the Diabetic Mouse

    ______________________________________                                                          Control                                                                              Treated                                              ______________________________________                                        EXPT I   Blood glucose (mmol/l)                                                  pre-dose 15.5 ± 3.7   15.6 ± 4.0                                        post dose (13 days) 34.0 ± 7.9 ***11.0 ± 3.6                            Blood glucose (mmo/l)                                                         pre-dose 16.5 ± 2.9   16.2 ± 4.3                                        Day 1 17.2 ± 3.7   14.1 ± 3.7                                          EXPTII Day 2 19.9 ± 2.7  **15.0 ± 3.4                                    Day 3 20.2 ± 3.9 ***12.2 ± 5.1                                          Day 5 21.2 ± 1.9 ***11.3 ± 3.6                                          Day 7 23.4 ± 2.0 ***11.7 ± 3.7                                       ______________________________________                                         Results are mean ± SD (n = 9-10 per group). ** p < 0.01; *** p < 0.001     vs controls.                                                             

Effect of(S)-3-[4-[2-[N-(2-Benzoxazolyl)-Nmethylamino]ethoxy]phenyl]2-(2,2,2-trifluoroethoxy)propanoicacid on Serum Lipids in the Genetically Diabetic Mouse

    ______________________________________                                                                 Treated (0.3 umol/kg                                   Parameter Control body wt)                                                  ______________________________________                                        Serum non-esterified                                                                         2.58 ± 0.45                                                                          ***1.28 ± 0.15                                      fatty acids (mmol/l)                                                          Serum triglycerides 3.78 ± 1.34 ***2.58 ± 0.45                          (mmol/l)                                                                    ______________________________________                                         Parameters were measured in samples taken after 14 days of treatment          (dietary admixture). Values are mean ± SD (n ± 9). *** p < 0.001        versus controls.                                                         

What is claimed is:
 1. A method for the treatment or prophylaxis ofSyndrome X in a human or non-human mammal, which method comprises theadministration of an effective, non-toxic and pharmaceutically effectiveamount of an agonist of PPARα and PPARγ, or a pharmaceuticallyacceptable derivative thereof, to a human or non-human mammal in needthereof.
 2. A method according to claim 1, wherein the agonist of PPARαand PPARγ is the same compound.
 3. A method according to claim 1 for thetreatment or prophylaxis of hyperglycemia.
 4. A method according toclaim 1, for the treatment of pre-diabetic insulin resistance syndromeand the resulting complications thereof.
 5. A method according to claim1, wherein the PPARα and γ agonist is a compound of formula (I):##STR33## or a pharmaceutically acceptable salt thereof, and/or apharmaceutically acceptable solvate thereof, wherein R⁰ represents2-benzoxazolyl or 2-pyridyl and R¹ represents CH₂ OCH₃ or CF₃.